BMPs, TGF-? and activin have been shown to have profound effects on bone mass in adults where most investigators have found that inhibiting canonical TGF-?/activin signaling enhances bone formation while inhibiting canonical BMP signaling decreases bone formation. However, an increasing number of studies have provided evidence to suggest that canonical BMP, TGF-? and activin signaling can have both positive and negative effects on osteoblast differentiation and function. As these molecules are often present in the extracellular environment at the same time, skeletal target cells are likely to receive inputs from both the TGF-?/activin and BMP canonical signaling pathways concurrently. Largely absent from most experiments where bone cells are treated with TGF-?/activin or BMPs is an examination of the potential intracellular antagonism that may occur between the two canonical signaling pathways. Studies focused on chondrocytes, another target cell that responds to TGF-?/activin and BMPs, provide intriguing data to suggest that these cells integrate signals from the two canonical pathways when making cell fate decisions. We hypothesize that there is bidirectional intracellular antagonism between the canonical BMP and TGF-?/activin signaling pathways in bone cells, and that the integrated signal that results from this interaction regulates bone mass in the adult skeleton. As existing reporters for BMP and TGF-?/activin signaling only give information about the independent activation of each pathway, and we need to determine the crosstalk between the pathways, we propose to construct a novel BMP/TGF-?/activin biosensor system that utilizes two independent reporters linked through reciprocal RNA interference. The inclusion of short hairpin RNA links the two reporters so that, through mutual antagonism, no fluorescence from either reporter will occur at a balanced signal but a fluorescent signal will occur when transduction through one pathway is dominant. Once validated in vitro, this biosensor will be used to map BMP vs. TGF-?/activin signaling in developing bone and in the adult skeleton in vivo. If successful, we anticipate that this biosensor will be useful to many investigators in multiple fields where an understanding of the balance between BMP and TGF-?/activin signaling is required. As such we believe that the impact of this innovative approach to understanding canonical signaling warrants the risks inherent in developing this novel reagent. PUBLIC HEALTH RELEVANCE: Diseases associated with aging have a tremendous impact on the health and wellbeing of all US citizens. This is particularly true for the skeleton where loss f bone in older adults threatens their independence. To better understand how to maintain bone mass as we age, we propose to develop a novel biosensor that allows us to assess if variation in levels of BMP/TGF-?/activin, signals that regulate bone formation, are responsible for age-related bone loss.